The increasing prevalence of antibiotic-resistant strains of bacterial pathogens represents a major unmet medical need. Naturally occuring or intentionally engineered drug-resistance in biothreat agents is also a serious risk for biodefense. The development of new antibiotics against unexploited targets with novel mechanisms of action is a vital part of the solution to these problems because such antibiotics will not be affected by pre-existing resistance alleles. Two essential components of the DNA replication pathway, helicase and primase, which act early and catalyze a rate-limiting step in replication, are currently untargeted by antibacterials. The goal of this project is to discover new inhibitors of the helicase or primase activities in bacterial DNA replication and develop them into antibiotics for biodefense and clinical applications. In Phase I research, active B. anthracis helicase and primase were purified and used to develop high-throughput inhibitor screens that were applied to a library of >180,000 discrete small molecule compounds and purified natural products. Evaluation of the hits in confirmation and validation assays identified four novel helicase inhibitors with potency vs. both S. aureus and B. anthracis helicases and one new primase inhibitor. These compounds exhibited sufficient antibacterial activity (MIC =25 ?g/ml) and selectivity for bacteria (CC50/MIC =2) to warrant further optimization. The most potent (MIC ~3 ?g/ml) and selective (CC50/MIC >16) inhibitor shares the bicyclic ring of the clinically-proven aminocoumarin scaffold;however, it does not inhibit DNA gyrase or the binding of ATP to helicase. Instead, it displays a mixed mode of inhibition including a component of competitive inhibition with the DNA substrate (Ki = 8 ?M). The inhibitor selectively inhibits DNA synthesis in macromolecular synthesis assays and is rapidly bactericidal at 4x MIC. The antibacterial spectrum as well as the observation that some inhibitors are nearly equipotent against B. anthracis and S. aureus helicases suggest that inhibitors can be optimized as broad-spectrum agents active against a range of Gram-positive bacterial species. In Phase II, analogs of the coumarin-type priority hit series will be designed, synthesized, and evaluated to optimize lead compounds through a drug discovery program incorporating rational design (SAR) and structure- based design elements. Analog design will be driven by determinations of potency, mechanism of action, antibacterial spectrum, and selectivity. The most promising lead compounds will be evaluated for favorable ADME properties and examined for pharmacokinetic properties, toxicity, and efficacy in animals to generate in vivo validated pre-clinical candidates. In Phase III, a potent, safe, orally active helicase/primase inhibitor will be advanced into IND-enabling toxicology and safety pharmacology studies, and an IND will be filed. PUBLIC HEALTH RELEVANCE: The increasing prevalence of antibiotic-resistant strains of bacterial pathogens is a concern both for biodefense and for combating ordinary infectious disease in the clinic. New antibiotics targeting previously unexploited bacterial functions will not be subject to most existing resistance mechanisms. The focused drug discovery and development effort of this proposal is aimed at optimizing novel inhibitors of DNA helicase and primase, which are vital for bacterial DNA replication. This research will provide new chemical classes of antibacterial therapeutics for biodefense and clinical applications.